Abstract
Although quite common in the eukaryotic cell, bacterial proteins with an extensive coiled-coil domain are still relatively rare. One of the few thus far documented examples, TlpA from Salmonella typhimurium, is characterized by a remarkably long (250 amino acids) alpha-helical coiled-coil domain. Herein, we demonstrate that TlpA is a novel sequence-specific DNA-binding protein. Several tlpA deletion mutants have been constructed, and their corresponding protein products were purified and tested for DNA binding. Two of the mutant proteins were shown to be deficient in DNA binding. Both mutants were analyzed by circular dichroism and electron microscopy, supporting the notion that mutant proteins wre shown to be deficient in DNA binding. Both mutants were analyzed by circular dichroism and electron microscopy, supporting the notion that mutant proteins were largely intact despite lacking the amino acid residues necessary for DNA binding. In vivo studies with transcriptional tlpA-lacZ fusions demonstrated that TlpA acts as a repressor. Using the repressor phenotype as a readout, the chain exchange previously described in vitro could also be confirmed in vivo. We believe the coiled-coil domain acts not only as a dimerization interface but could also serve a role as a flexible modulator of the protein-DNA interaction.
Highlights
Quite common in the eukaryotic cell, bacterial proteins with an extensive coiled-coil domain are still relatively rare
Two of the mutant proteins were shown to be deficient in DNA binding
Both mutants were analyzed by circular dichroism and electron microscopy, supporting the notion that mutant proteins were largely intact despite lacking the amino acid residues necessary for DNA binding
Summary
Plasmid Construction—Methods for DNA manipulation and transformation have been previously described [23]. PMR12 and p3062d5 were manufactured by replacing in tlpA the region of codons 31 to 371 with a PCR1-generated fragment encoding residues 43–371. The pOF tlpA-lacZ transcription fusion constructs were based on the pACYC184 vector (New England Biolabs) containing a lacZ cartridge in the BamHI-SalI sites (pKTH3090) [24]. The truncated tlpA region that regulates lacZ expression in pOF6 was generated by PCR using S. typhimurium pEX102 virulence plasmid DNA as a template. From the resultant plasmids the inserts were cut out as BamHI-HindIII fragments before ligation into the corresponding cloning sites of pKTH3090. Each binding reaction contained about 15,000 cpm labeled fragments in gel mobility shift binding buffer at final NaCl concentration of 150 mM. The DNA used for footprinting was the 223-bp fragment produced by PCR with either one of the two oligonucleotides carrying the radioactive label. The specimens were examined in a Jeol 1010 microscope operated at 100 keV
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
